Part:BBa_K4990011

Bacteria-Targeting Rod

Usage in short

You can use it to achieve BL-Fn adhesion.

You need to know

Acurate targeting is one important characteristics for many emerging cancer therapies, that is also what ourproject’s ambition. We designed two fusion proteins, they function like fishingrod, so we gave them the name: Fishing Rod Proteins (FRPs). The one that targetFn names Bacteria Tageting Rod(BTR), the other that target CRC cells names Tumor Tageting Rod(TTR). FRPs are displayed on engineered BL membranes, helping BLtarget to the correct place. Of course, their targets are cancer cells and Fn,rather than fish. Each FRP consists of four parts: the signal peptide(white),helping FRPs locate on cell membrane; the membrane protein(violet), the base of FRPs; and the linker(yellow), helping FRPs become flexible; and the targeting fragments(Orange), the most pivotal role in FRPs

What is it

It consists of the GL-BP membrane protein,GSGSG flexible linker, and B-domain. Essentially, it involves the surface display technique to express B-domain on the BL membrane for adhesion to FadA, thereby achieving adhesion between BL and Fn.

What it can do

the targeting fragment of BTR canbind to Fn, but its mechanism is more special. FadA is Fn’s pilus and composed of a number of mFadA monomers. The monomers connect to each other byself-assemblely,and that is the point of our design. The binding force between mFadA was strong,so we intercepted peptides that interact with monomers as our targeting fragments.Actually, the binding of Fn and BTR is the self-assembly process with the mFadA monomer.All in all, with the guidance of TTR and BTR,engineered BL is able to find CRC or Fn. Although some Fn may not have reached the tumor, since Fn can adhere to CRC cells, all BL will located in TME ultimately.

The B-domain of mFadA is our target bacterial fragment, and we explored the bacteria targeting ability of BTP and DEH by validating the interaction between the B-domain and mFadA.In lanes 6-8, clear bands appears around 30kDa suggesting that DEH (~15kDa) can bind with pili monomers via interaction with mFadA (~15kDa). This proves that DEH can target pathogenic bacteria Fn by participating in the self-assembly process of pilis (Fig 6C(ii)). Furthermore, the self-assembly phenomenon of mFadA may result in ladder-like bands at different molecular weight positions. These bands could be due to the interaction of the BTP-mFadA-mFada complex (~35kDa,Fig 6D(i)), and the BTP-mFadA-mFadA-mFadA complex (~50kDa, Fig 6D(ii)). Similarly, the breakage of the linker in DEH and the dimerization of HlpA can lead to a more complex band situation. The unexpected band could also formed due to HlpA-DEH-mFadA complex(~40kDa, Fig 6E(i)),DEH-mFadA-mFadA complex(~45kDa, Fig 6E(ii)), DEH-mFadA-mFadA-mFadA complex(~60kDa, Fig 6E(iii)) etc. These unexpected bands have indirectly verified the self-assembly of pili monomers and the dimerization of HlpA, thereby providing further evidence for the reliability of our project.

The interaction between BTP and mFadA, or between DEH and mFadA.|A. BTP-mFadA complex (~20kDa, C(i)) and DEH-mFadA complex(~30kDa(C(ii)) could be observed (red squares). And bands at other different molecular weight position appear due to the self-assemble of mFadA and the dimeration of HlpA in DEH (blue squares), whose structures are annotated beside.B. The structure of BTP.C. The interaction between BTP and mFadA(C(i)), or between DEH and mFadA(C (ii))D&E. The complexes formed due to the self-assembly of mFadA(D) and the dimerization of HlpA(E).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 358
  Illegal PstI site found at 1450
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 358
  Illegal PstI site found at 1450
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 771
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 358
  Illegal PstI site found at 1450
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 358
  Illegal PstI site found at 1450
  Illegal NgoMIV site found at 1050
  
• 1000
  COMPATIBLE WITH RFC[1000]